Metastasis is a major cause of death of breast cancer (BCa) patients. Angiogenesis is a necessary step for breast cancer growth and progression. Signaling via vascular endothelial growth factor (VEGF) plays a vital role in tumor angiogenesis. Our working hypothesis is that p16 downregulates VEGF gene expression at the transcriptional level, thus inhibiting angiogenesis, which contributes to p16-mediated suppression in BCa growth and metastasis. AIM 1: To elucidate the mechanism by which p16 downregulates the VEGF signaling pathway. Aim 1.1. To confirm that p16 downrequlates VEGF at both mRNA and protein levels in BCa cells. Aim 1.2. To determine whether p16 downregulates VEGF gene expression at the transcriptional level. Aim 1.3. To determine the molecular mechanism by which p16 downregulates VEGF expression at the transcriptional level. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator for the VEGF gene promoter. HIF-1 is composed of an inducible subunit, HIF-1alpha and a constitutively expressed subunit, HIF-1beta. Heterodimerization of HIF-1alpha and HIF-1beta is required to form the transcriptionally active HIF-1. We hypothesize that p16 downregulates VEGF gene expression at the level of transcription. Binding of p16 to HIF-1alpha prevents HIF-1 transcriptional activity via decreased HIF-1alpha interaction with HIF-1beta. We will use the following three subaims to test this hypothesis. 1.3.1. To determine whether p16 directly binds to HIF-1alpha and alters HIF-1alpha cellular localization. 1.3.2. To determine whether p16 blocks (i) the formation of the HIF-1alpha and HIF-1beta complex and (ii) HIF-1 binding activity to HRE (HIF-1 binding site) DNA region. 1.3.3. To determine whether p16 specifically inhibits HIF-1alpha-induced VEGF transcription. Aim. 1.4. To determine whether p16 affects VEGF receptor expression and phosphorvlation. AIM 2: To determine p16's effects in suppression of breast tumor growth, angiogenesis and metastasis. Aim. 2.1. To determine whether p16 inhibits BCa cell growth in vitro. Aim. 2.2. To determine whether p16 inhibits BCa-induced angiogenesis. The effects of p16 on angiogenesis will be evaluated in both in vitro and in vivo angiogenic assays. Aim. 2.3. To determine whether p16 suppresses breast tumor metastasis in an in vivo model. BCa cells stably transfected with Tet-on regulated p16 expression vector will be administrated into mice by mammary fat pad injection (spontaneous metastasis model) and by tail vein injection (experimental metastasis model). The effects of inducible p16 expression on suppression of primary tumor growth and secondary tumor formation (lung metastases) will be evaluated in both models. The long-term objectives are (I) to clarify the mechanism of how p16 downregulates VEGF gene expression;and (II) to determine whether inhibition of tumor angiogenesis via downregulation of VEGF by p16 is an effective way to suppress BCa growth and metastasis.